Malaysian Journal of Microbiology, Vol. 11(4) 2015,pp. 358-364 http://dx.doi.org/10.21161/mjm.71415 Malaysian Journal of Microbiology Published by Malaysian Society for Microbiology (In since 2011)

Isolation and identification of lactic acid bacteria from different stages of traditional Malaysian production

1 1,2* 1 3 Balqis Pisol , Noriham Abdullah , Khalilah Abdul Khalil and Lilis Nuraida

1Faculty of Applied Science, Universiti Teknologi MARA, 40450 Shah Alam, Selangor, . 2 Malaysia Institute of Transport (MITRANS), Universiti Teknologi MARA, 40450 Shah Alam, Selangor, Malaysia 3Department of Food Science and Technology, Faculty of Agricultural Technology, and SEAFAST Center, Bogor Agricultural University, IPB Darmaga Campus, PO BOX 220, Bogor, West Java, . Email:[email protected]

Received 4 March 2015; Received in revised form 20 May 2015; Accepted 28 May 2015

ABSTRACT

Aims: There are many methods of soybean tempeh production as they vary according to the producers. Isolation of lactic acid bacteria (LAB) from tempeh was carried out at different stages of the tempeh production to examine the occurrence of LAB and to identify the isolates. Methodology and results: Morphological, physiological and chemical characteristics with the use of API 20 Strep, API 50 CHL kit and 16S rRNA gene sequences were employed to identify LAB. By using API 20 Strep and API 50 CHL kit, fourteen LAB were obtained and twelve isolates have been successfully identified: seven coccus LAB isolates as Enterococcus faecium, four cocci-bacilli as Leuconostoc mesenteroides ssp. mesenteroides, one bacilli as Lactobacillus delbrueckii ssp. delbrueckii. Meanwhile, two bacilli isolates were categorised as unidentified strain. On the other hand, molecular identification using 27f and 1429r primer had revealed that L. mesenteroides and L. delbrueckii were identified as Leuconostoc lactis and Leuconostoc sp. respectively. Whereas, two previously unidentified bacilli isolates were identified as Alicyclobacillus sp. Conclusion, significance, and impact of study: This result shows that various types of LAB was detected at every stages of tempeh production and had been identified by using two different techniques. The unique characteristics of LAB offer their potential towards food and pharmaceutical industry.

Keywords: Soybean, tempeh, lactic acid bacteria, isolation, identification.

INTRODUCTION advantageous influence on nutritional values, organoleptic, and shelf-life characteristics of the fermented food (Leroy Tempeh is a patty of cooked soybean being fermented by and De Vuyst, 2004). Occurrence of lactic acid bacteria mould starter culture Rhizopus spp., most commonly (LAB) have been investigated from tempeh and other Rhizopus oligosporus. Soybean tempeh is a famous famous Malaysian fermented foods such as , fermented food in Malaysia and this food gains its , and cincalok (Kormin et al., 2001; Moreno et popularity from its taste, digestibility, nutritional value and al., 2002; Liasi et al., 2009; Belal and Zaiton, 2011; well-known as high-quality meat replacers (Nout and Kalavathy et al., 2012; Hajar and Hamid, 2013). Ability of Aidoo, 2010). Yellow seeded soybean (Glycine max) is these microorganisms to produce variety of antimicrobial the most preferred type, use as the raw material although compounds and combat pathogenic microorganisms many other types of soybean and legumes can be used (Menssens and De Vugst, 2002), makes them bacteria of such as red kidney bean (Srapinkornburee et al., 2009), interest for probiotic purposes. mucuna (Sri Handajani, 2001), chickpea (Abu-Salem and In this study, the method used for tempeh production Abou-Arab, 2011), and barley (Feng, 2006). is different than most of the methods described by other The group of lactic acid bacteria (LAB) is responsible authors as this method involved double soaking process. to accelerate the fermentation process of tempeh. LAB The soaking process is very important as it functions to are Gram positive, bacilli and cocci shape, catalase increase the moisture content of the beans, make the negative and produce lactic acid as their main product. beans edible, facilitate microbial activity during the Aside from being well known for its GRAS (Generally fermentation, and to naturally extract antimicrobial Regarded As Safe) status, this type of microorganisms (saponins) and bitter compounds (Nout and Aidoo, 2010). has a long history of application prior to their Although additional time is required, soaking process is

*Corresponding author

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still the only inexpensive and simple method considering carbonate (CaCO3) using pour plate method and were its multi-effects in enhancing nutritional value and lowering incubated for 48 h at 37 °C. The identified colonies were the anti-nutritional factor (Mumba et al., 2004). This counted and total numbers of viable cells were reported includes significant reduction of phytate (Karkle and as colony-forming units (CFUs). White small colonies with Beleia, 2010), increase of total soluble solid (Arbianto, clear zone around them were randomly isolated using 1978) and decrease of oligosaccharides, sucrose content sterilized toothpicks and streaked on fresh MRS . and also tannins (Egounlety and Aworh, 2003). Other steps of tempeh production are the same as the method used by most producers except some might varies in Soybean terms of incubation time, temperature and packaging materials. Hence this study was conducted with the aim to isolate LAB from tempeh production that involved both double soaking process and over-fermented tempeh. 1st day of soaking (24 h, RT) * Awareness on importance of healthy foods contributes to the high interest and demand for natural fermented foods.LAB has the advantages to offer the solution for this problem. However, the number of local LAB strains isolated from tempeh is still scarce. As a result, isolation Cooking (10 min, 100°C) of LAB from local resources like Malaysian tempeh is necessary. The aim of this study is to isolate and identify the natural population of LAB from traditional tempeh processing. The significant of this study is to make use of nd local fermented product and its waste to its best use. 2 day of soaking (24 h, RT) * Efforts to understand LAB isolated from local fermented food may be of value to the Malaysian economy.

MATERIALS AND METHODS Cooking (30 min, 100°C) Tempeh production

Production of soybean tempeh was prepared according to a local producer in Klang, Malaysia. Yellow seeded soybean (Country Farm Organics) was used to make the Inoculation of inoculums at RT (Day 0) tempeh. In this study, 100 g soybean was soaked in 1 L water for 24 h at room temperature (RT) and then drained. It was then cooked at 100 °C for 10 min in 1 L water. After boiling, half cooked soybean was left soaked in the boiled water and left for another 24 h at RT. Next, the water was Tempeh (Day 2)* drained and washed several times to remove the hulls. Soybean was further cooked at 100 °C for another 30 min and the water was drained subsequently. The cooked soybean was cooled at RT for 20 min and then mixed Incubation thoroughly with 0.03 g inoculums (Raprima Inokulum Tempe (Day 4)* at 37 °C Tempeh, PT Aneka Fermentasi Industri, , Indonesia). Finally, soybean was packed into perforated plastic bag and incubated at 37 °C for 7 days. The overall production of soybean tempeh and stages where samples were taken is shown in Figure 1. All samples were freshly Tempe (Day 7)* analysed on the same day they were produced.

Isolation of LAB Figure 1: Flowchart of tempeh production (* Stage where sample was taken). Each sample of 25 g was aseptically added into 225 mL of sterile peptone water (0.1% w/v) solution and was mixed Purification thoroughly. Isolation of LAB was performed as method described by Muhialdin and Hassan (2011) with slight Single colony was picked from the plates and subculture modification. Serial dilutions were performed and 1 mL to purify. Pure bacterial strains were obtained after five aliquots of appropriate dilution were directly inoculated in times successive transfer of individual colony on the duplicate on the de Man Rogosa Sharpe (MRS) agar plates and incubated for 24 h at 37 °C. Culture (Oxoid Ltd, Basingstoke, UK) with 0.8% calcium preservation was performed on all the isolates obtained.

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Fresh overnight cultures of each isolate were grown in (http://www.ncbi.nlm.nih.gov). A definite confirmation of broth and stored in the freezer at −80 °C in 20% glycerol the strain was obtained on the basis of these results. (Kheadr, 2006). RESULTS AND DISCUSSION Identification of LAB Isolation and purification of LAB Identification of LAB was done based on morphology and catalase reaction. Morphological observations of LAB with After the purification process, a total of 14 strains of LAB gram staining were carried out. Based on previous which produce clear zone around their colonies were description, it is known that LAB is gram-positive with rod isolated from different stages of tempeh production. The or cocci shape. Catalase test was performed using freshly appearance of clear zone was due to dissolution of prepared 3% hydrogen peroxide (H2O2). CaCO3 in MRS medium by acid production. All isolates were Gram positive, catalase negative and have the Identification of LAB using API 20 Strep and API 50 shape of bacilli, cocci and cocci-bacilli. From the isolation CHL process, it was observed that LAB present at all stages of tempeh production (Table 1). All isolates with gram positive and catalase negative characteristic were tested for the production of acids from Table 1: Enumeration of LAB at different stages of carbohydrates and related compounds by using API 50 tempeh production. CHL and API 20 Strep (API System, Bio-Merieux, France). The API 50 CHL system was used for the identification of Number of LAB at different Lactobacillus and related genera while API 20 Strep was Stage stage of tempeh production used for the enzymatic and carbohydrate fermentation (log CFU/mL) patterns of most streptococci and enterococci strains. All Day 1 soaking water 7.35-7.56 tests were conducted according to the manufacturer’s Day 2 soaking water 7.40-7.79 instructions. All data were obtained from the kit after Tempeh (day 2) 8.11-8.92 incubation for a period 4 to 48 h. Data were recorded and Tempeh (day 4 ) 7.34-8.45 analysed using API LAB Plus, version 5.1. Interpretations Tempeh (day 7) 7.74-8.00 of the respective bacterium were compared with The values are expressed as minimum and maximum. information from the database. From Table 1, it was observed that the number of LAB Identification using 16S rDNA from day 1 soaking water were in the range of 7.35-7.56 log CFU/mL. It is in agreement with the range of 7.9- 9.3 All 14 LAB isolates were identified using 16S rRNA gene log CFU/mL as reported by Moreno et al. (2002). As for sequencing. LAB isolates were initially grown in MRS day 2 soaking water, the number of LAB were in the range broth for 24 h at 37 °C. The DNA was extracted using the of 7.40- 7.79 log CFU/mL. This showed that the number of DNeasy Blood and Tissue kit (QIAGEN, German), LAB increased with the time of soaking. According to according to the manufacturer’s instructions. The 16S Nuraida et al. (2008) the value of LAB was decreased (- rRNA gene was amplified with universal primers 27f (5’- 1.63 log CFU/mL) from soaking water from day 1 to day 2 AGAGTTTGATCATG CCTCAG -3’) and 1429r (5’- while in this study the number of LAB was slightly TACGGTTACCTTAC CTTGTTACGACTT-3’) (Kalavathy increased (+ 0.14 log CFU/mL). The study also reported et al., 2012). Amplification was performed in a 25 µL that as time increased from soaking day 1 to day 2, the pH volume containing 12.5 µL TopTaq Master Mix (QIAGEN, of soaking water became more acidic with changes from German), 9.5 µL dH2O, 10 µM 27f primer, 10 µM 1429r pH 5.25 to pH 4.84 (Nuraida et al., 2008). The number of primer and 10 µM template DNA. Polymerase chain LAB at all stages was between 7.34-8.92 log CFU/mL reaction (PCR) was run using Bio-Rad, MJ Mini Thermal throughout the whole process. This finding was also in Cycler under following conditions: one cycle of initial agreement with Moreno et al. (2002) and Balqis et al. denaturation at 95 °C for 5 min, followed by 30 cycles of (2013) who reported that the numbers were in the range denaturation at 94 °C (45 sec), annealing at 47 °C (30 of 6.8-9.9 and 6.88-8.98 log CFU/mL respectively. The sec), elongation at 72 °C (45 sec) and final extension at numbers of LAB obtained in this study was in agreement 72 °C for 5 min (Kalavathy et al., 2012). The PCR with findings by Liu et al. (2012) who reported that the products were loaded in 0.7% agarose gels for range was between 6.0-9.3 log CFU/mL. Occurrence of electrophoresis and visualised by GelStar staining LAB at all stages of tempeh making is very important (Lonza, USA) under UV transilluminator. The PCR because during soaking and fermentation, growth of LAB products were sent for purification and sequencing to My helps in acidifying the soaking water and tempeh itself TACG Bioscience Enterprise (Selangor, Malaysia). which in turn inhibits the growth of potential pathogenic Sequences were manually edited and aligned using DNA bacteria (Nout et al., 1987; Ashenafi and Busse, 1992). Baser version 4.16.0 and matched with DNA sequences The presence of yeast and mould were also observed from GenBank using the BLAST software (BLASTN) at at all stages of tempeh processing except during soaking. the National Centre of Biotechnology Information, NCBI Isolation by Moreno et al. (2002), also reported the

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presence of mould and yeast at different stages of tempeh Staphylococcus spp. even though the count was below production although they were cultured on different media. the numbers that could cause food poisoning. Some of the This finding was also supported by Balqis et al. (2013) bacteria reported to be obtained from over-fermented who reported that the presence of mould in the final tempeh were; Escherichia coli, Enterobacter aerogenes, product was predicted since the mould was intentionally Staphylococcus, Vibrio cholerae, Proteus morganii and P. incorporated into the tempeh as an inoculum. vulgaris (Fardiaz et al., 1990). It was observed that as the fermentation time increased, the number of LAB present also increased Identification of LAB except for tempeh at day 4 as the sample had already spoiled and dominated by spoilage causing bacteria. The Gram staining technique is a very crucial test as it will Research by Fardiaz et al. (1990) on over-fermented assist in identifying the LAB. Only Gram positive, rod and soybean tempeh excellently discussed on the physical cocci shape bacteria were selected and tested for and chemical changes of tempeh during the prolonged catalase. Therefore, as many as 14 bacteria isolates were fermentation. In his work, it was proven that there was used for further identification (Table 2). significant increase in gram negative bacteria and

Table 2: Isolation and identification of LAB.

Source Number of Gram stain Catalase Morphology API identification isolate 6 + - Cocci A, B, C, D, E, F- Enterococcus Day 1 soaking water faecium 2 + - Cocci-bacilli G, H- Leuconostoc mesenteroides Day 2 soaking water ssp. mesenteroides 2 + - Cocci 1-Enterococcus faecium Tempeh (day 2) Bacilli J-Lactobacillus delbrueckii ssp. delbrueckii Tempeh (day 4 ) 1 + - Bacilli K- Unidentified 3 + - Bacilli L- Unidentified Tempeh (day 7) Cocci-bacilli M, N- Leuconostoc mesenteroides ssp. mesenteroides

Identification of LAB using API 20 Strep and API 50 was performed. All PCR products were shown to produce CHL 1.5 kb fragment (Figure 2). The BLAST result analysis had revealed that Leuconostoc mesenteroides and L. A total of 12 strains out of 14 isolates from different stages delbrueckii were reidentified as Leuconostoc lactis (100% of tempeh production were satisfactorily identified using identity) and Leuconostoc sp. (99% identity), respectively. API 20 Strep and API 50 CHL kit as shown in Table 2. API The unidentified isolated were identified as 20 Strep was used for the enzymatic and carbohydrate Alicyclobacillus sp. (100% identity). The remaining LAB fermentation for enterococci strains. Seven cocci isolates isolates were confirmed as Enterococcus faecium with 99- were excellently identified as Enterococcus faecium with 100% identity (Table 3). % ID between 99.3-99.7%. All bacilli and cocci-bacilli Based on the results obtained, it was clearly shown shape bacteria were identified using API 50 CHL which that there was significant different in types of LAB from was identified based on carbohydrate fermentation with day 1 and day 2 soaking water. As for day 1 soaking respect to sugar utilisation and acid production of each water, the type of LAB dominating this sample was the species. Four other isolates were identified as cocci shaped LAB identified as Enterococcus faecium Leuconostoc mesenteroides ssp. mesenteroides (% ID: while day 2 soaking water was dominated by cocci-bacilli 95.7-99.9%), one isolates as Lactobacillus delbrueckii ssp. shaped LAB identified as Leuconostoc sp. and delbrueckii (% ID: 97.8%).However, two isolates from Leuconostoc lactis. tempeh at day 4 and day 7 could not be identified with the Absence of Enterococcus faecium from day 2 soaking result appeared as doubtful profile. According to Bill et al. water may be due to the boiling temperature during (1992) and Klinger (1992), some commercial identification cooking of the soybean. Generally, the species identified systems often yield good result with respect to genus but in this study were in good agreement with other work by not sufficient at species level. previous researchers. Kalavathy et al. (2012), isolated Lactobacillus sp. and Pediococcus acidilactaci from Identification using 16S rDNA tempeh with the antibacterial properties against Micrococcus luteus. Meanwhile Belal and Zaiton (2011) For more accurate result, identification at molecular level

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Mal. J. Microbiol. Vol 11(4) 2015, pp. 358-364 discovered Lactobacillus brevis and Pediococcus faecalis were LAB isolated from similar fermented pentosaceus whereas Kormin et al. (2001) identified soybean, douchi (Liu et al., 2012). Findings from this Lactobacillus plantarum BS2 from tempeh also with study were also supported by Balqis et al. (2013), with the similar characteristics. Furthermore, Enterococcus identification of heterofermentative Lactobacillus spp. at faecium was successfully obtained from 7 days old different stages of tempeh production along with fermented tempeh (Moreno et al. 2002). Lactobacillus Streptococcus non-enterococci bacteria from 2 days old spp., Weisella spp., Pediococcus spp. and Enterococcus tempeh.

Table 3: Identification of LAB isolates using BLAST software.

Isolate Species Identity (%) Accession number A Enterococcus faecium 99 CP006030.1 B Enterococcus faecium 99 CP006030.1 C Enterococcus faecium 99 KJ728981.1 D Enterococcus faecium 100 KJ728981.1 E Enterococcus faecium 99 CP006030.1 F Enterococcus faecium 99 CP006030.1 G Leuconostoc lactis 100 AB904777.1 H Leuconostoc sp. 99 JX026036.1 I Enterococcus faecium 99 KJ728981.1 J Leuconostoc sp. 99 JX026035.1 K Alicyclobacillus sp. 100 KC893645.1 L Alicyclobacillus sp. 100 KC893647.1 M Leuconostoc lactis 100 KJ095665.1 N Leuconostoc lactis 100 KJ095665.1

3000

2000

1500

Figure 2: The PCR amplification of ribosomal RNA gene using 27f and 1429r primer. Lanes 2-16 corresponds to genomic sample of the LAB isolates except lane 7. Lane 1 is control in this case is L. plantarum.

CONCLUSION Enterococcus faecium (7), Leuconostoc lactis (3), Leuconostoc sp. (2) and Alicyclobacillus sp. (2). Day 1 In this study, LAB has been successfully isolated from soaking water was dominated by cocci shape LAB different stages of tempeh production with 50% cocci identified as Enterococcus faecium while day 2 soaking shape, 35.7% cocci-bacilli shape and 14.3% bacilli shape water was dominated by cocci-bacilli shaped LAB LAB. Overall, all 14 LAB was successfully identified as: identified as Leuconostoc sp. and Leuconostoc lactis. This

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Mal. J. Microbiol. Vol 11(4) 2015, pp. 358-364 shows that different types of microorganisms were Feng, X. M. (2006). Microbial dynamics during barley involved during prolonged soaking and tempeh production. tempeh fermentation. Ph.D. Thesis. Swedish LAB isolates obtained will be further characterised for University of Agricultural Sciences, Uppsala. ISSN probiotic purposes before it can be applied in food and 1652-6880, IBSN 91-576-7108-7. pharmaceutical products. Hajar, S. and Hamid, T. H. T. A. (2013). Isolation of lactic acid bacteria strain Staplylococcus piscifermentas ACKNOWLEDGEMENTS from Malaysian traditional fermented cincaluk. International Food Research Journal 20(1), 125-129. This study was supported by the collaboration of Universiti Karkle, E. N. L. and Beleia, A. (2010). Effect of soaking Teknologi MARA (UiTM), Malaysia with Institut Pertanian and cooking on phytate concentration, minerals, and Bogor (IPB), Indonesia and funded by the Research texture of food-type soybeans. Ciência e Tecnologia Cluster Grant [600-RMI/ DANA 5/3/CG (18/2012)] and de Alimentos 30(4), 1056-1060. Fundamental Research Grant Scheme (FRGS) [600-RMI/ Kalavathy, R., Nor Zaihana, A. R., Sieo, C. C., FRGS 5/3 (147/2013)]. NurJahan, A., Norhani, A. and Ho, Y. W. (2012). Probiotic potential of lactic acid bacteria from REFERENCES fermented Malaysian food or milk products. International Journal of Food Science and Technology Abu-Salem, F. M. and Abou-Arab, E. A. (2011). 47, 2175-2183. Physico-chemical properties of tempeh produced from Kheadr, E. E. (2006). Impact of acid and oxgall on chickpea seeds. Journal of American Science 7(7), antibiotic susceptibility of probiotic Lactobacilli. African 107-118. Journal of Agricultural Research 1(5), 172-181. Arbianto, P. (1978). 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